Analysis of anemia and iron supplementation among glioblastoma patients reveals sex-biased association between anemia and survival

The association between anemia and outcomes in glioblastoma patients is unclear. We analyzed data from 1346 histologically confirmed adult glioblastoma patients in the TriNetX Research Network. Median hemoglobin and hematocrit levels were quantified for 6 months following diagnosis and used to classify patients as anemic or non-anemic. Associations of anemia and iron supplementation of anemic patients with median overall survival (median-OS) were then studied. Among 1346 glioblastoma patients, 35.9% of male and 40.5% of female patients were classified as anemic using hemoglobin-based WHO guidelines. Among males, anemia was associated with reduced median-OS compared to matched non-anemic males using hemoglobin (HR 1.24; 95% CI 1.00–1.53) or hematocrit-based cutoffs (HR 1.28; 95% CI 1.03–1.59). Among females, anemia was not associated with median-OS using hemoglobin (HR 1.00; 95% CI 0.78–1.27) or hematocrit-based cutoffs (HR: 1.10; 95% CI 0.85–1.41). Iron supplementation of anemic females trended toward increased median-OS (HR 0.61; 95% CI 0.32–1.19) although failing to reach statistical significance whereas no significant association was found in anemic males (HR 0.85; 95% CI 0.41–1.75). Functional transferrin-binding assays confirmed sexually dimorphic binding in resected patient samples indicating underlying differences in iron biology. Anemia among glioblastoma patients exhibits a sex-specific association with survival.


Classification of anemia and quantification of comorbidities and therapeutics
Logical Observation Identifiers, Names and Codes (LOINC) or TriNetX (TNX) codes were used to identify hemoglobin, hematocrit, mean corpuscular volume, and white blood cell count lab values in each patient's record from the day of diagnosis to 6 months afterward (Supplementary Table 8).Median hemoglobin and hematocrit levels were calculated for each patient for this time period and used to classify patients as anemic or non-anemic.World Health Organization hemoglobin cutoffs for anemia of 13 g/dL for male and 12 g/dL for female patients were used 25 .Hematocrit cutoffs of 39% for male and 36% for female patients were used as alternate criteria for anemic classification.Comorbidities before glioblastoma diagnosis were identified using ICD-9 and ICD-10 codes (Supplementary Table 6) in the patient record, and a Charlson Comorbidity Index for each patient was calculated using the comorbidity prevalence as described previously 26 .Patients were identified as having received chemotherapy using RxNorm codes for temozolomide, carmustine, or lomustine.Anemic patients were identified as having received an iron supplement using RxNorm codes for a panel of iron compounds (Supplementary Table 7).Patients were considered to have been iron supplemented if the iron supplement was received within 5 years before or 6 months after diagnosis.

Statistical analysis
All analysis was performed using R version 3.6.3.For the analysis of anemic status and association with median-OS by sex, to account for potential confounders, anemic versus non-anemic cohorts were balanced for the covariates of age at diagnosis, Charlson comorbidity index, whether patients received chemotherapy and/or radiation, time interval from diagnosis to initiation of chemotherapy and/or radiation, white blood cell counts, number of hemoglobin measurements (to account for possible differences in care received between those with a few measurements versus those with much higher measurements), and comorbidities using 1:1 propensity score matching with logistic-regression-based distance calculation and nearest-neighbor matching without replacement and with a caliper distance of 0.1 standard deviation of the logit propensity score using the MatchIt package in R 27 .Balance was assessed by examining the absolute standardized mean differences between the covariates of the anemic versus non-anemic cohorts using the cobalt package in R (Supplementary Fig. 1A-D) 28,29 .Balance was also assessed by statistical testing of the covariates before and after matching (Tables 1, 2, Supplementary Tables S1, S2).For the analysis of iron supplementation of anemic patients by sex, because of limited sample size, cohorts were balanced only for age at diagnosis, Charlson comorbidity index, and whether patients received Table 1.Patient characteristics anemic versus non-anemic female patients as defined using hemoglobin cutoffs of 12 g/dL.Columns on the left represent characteristics of the cohorts before propensity score matching and columns on the right represent characteristics of the cohorts after propensity score matching.Comparisons that were statistically significant using a threshold of P < 0.05 are in bold.HIV or AIDS 3 (0.9) 1 (0.4) 0.901 1 (0.5) 1 (0.5) 1 samples were minced and homogenized in 0.3 M sucrose (VWR Chemicals).Homogenates were centrifuged at 800 × g for 20 min to remove large debris.Supernatants were collected and further centrifuged at 120,000 × g for 1 h and resulting pellets were re-suspended in potassium phosphate buffer supplemented with 10% glycerol (Fisher-Scientific) to isolate plasma membrane extracts. 125I-tagged human transferrin (Sigma-Aldrich) was subsequently added to 20 µg of plasma membrane extract in a 96-well filter plate (Millipore-Sigma) and allowed to incubate for 1 h at room temperature.The reaction was then terminated by addition of ice-cold PBS and the mixture was subsequently vacuum filtered using 0.2 µm hydrophilic membranes.Filters were then washed three times with ice-cold PBS to remove unbound radiolabeled transferrin.Radioactivity in the filters was then quantified using a Beckman Gamma 4000 Analyzer.
Table 2. Patient characteristics anemic versus non-anemic male patients as defined using hemoglobin cutoffs of 13 g/dL.Columns on the left represent characteristics of the cohorts before propensity score matching and columns on the right represent characteristics of the cohorts after propensity score matching.Comparisons that were statistically significant using a threshold of P < 0.05 are in bold.

Number of patients (N)
Before

Anemia in male patients is associated with reduced median overall survival
The association of anemia with median overall survival (m-OS) was assessed after classifying patients as anemic using WHO based hemoglobin cutoffs of 12 g/dL for females and 13 g/dL for males.To account for baseline differences between the anemic and non-anemic patients, the two cohorts were balanced using propensity score matching.After matching, balance between anemic and non-anemic cohorts was assessed for age at diagnosis, Charlson comorbidity index, white blood cell counts, total number of hemoglobin measurements, whether patients received chemotherapy and/or radiation, time interval between diagnosis to initiation of chemotherapy and/or radiation, and systemic comorbidities.The cohorts were found to be acceptably balanced using the commonly accepted threshold of 0.1 absolute mean standardized difference between covariates (Supplementary Fig. 1A,B) or by statistical testing (Tables 1, 2, Supplementary Tables 1, 2).Survival between balanced anemic and non-anemic cohorts stratified by sex was then assessed using Kaplan-Meier curves, log-rank testing, and univariate Cox regression with robust variance estimation.No association was found between anemia and median-OS www.nature.com/scientificreports/ in females (median-OS 15.6 vs. 16.6 months, P = 0.989, HR 1.00; 95% CI 0.78-1.27)(Fig. 2A,B) but anemia was found to be associated with reduced median-OS in males (median-OS 12.8 vs. 16.0 months, P = 0.049, HR 1.24; 95% CI 1.00-1.53)(Fig. 2C,D).
As an alternate to hemoglobin-based guidelines of anemia, hematocrit was used as the parameter for anemia classification.A hematocrit of 39% was defined as the anemia cutoff for males and 36% for females.After balancing cohorts as described above, balance was assessed and verified to be acceptable for all covariates apart from smoking status and rheumatic disease in males which had SMD slightly greater than 0.1 but acceptable balance on statistical testing (Supplementary Fig. 1C,D and Supplementary Tables 1 and 2).Sex-stratified Kaplan-Meier analysis, log-rank testing, and univariate Cox regression with robust variance estimation again revealed no association of anemia and median-OS in females patients (median survival 14.9 vs. 17.9 months, P = 0.476, HR 1.10; 95% CI 0.85-1.41)(Fig. 3A,B) but a strong association of anemia and reduced median-OS in males (median-OS 13.0 vs. 16.8 months, P = 0.025, HR 1.28; 95% CI 1.03-1.59)(Fig. 3C,D).

Figure 2.
Sex-specific overall survival of patients stratified by anemic versus non-anemic using hemoglobin cutoffs.Sex-specific median overall survival was assessed for anemic versus non-anemic patients before (A, C) and after (B, D) propensity score matching.Anemia, as defined using World Health Organization cutoffs of < 12 g/dL for females and < 13 g/dL for males, was associated with reduced 5-year overall survival.

Iron supplementation is associated with a trend toward improved survival in anemic female patients
We next investigated whether iron supplementation of anemic patients may be associated with survival.Among 280 anemic males, 29 (10.4%) were found to have received an iron supplement.Among the 229 anemic females, 34 (14.8%) received an iron supplement.The iron supplemented and non-supplemented patient cohorts were then stratified by sex and balanced for age at diagnosis, Charlson score, and whether patients received chemotherapy and radiation using propensity score matching to reduce bias.Because of limitations in sample size, balance was assessed only for age at diagnosis, Charlson comorbidity index, and whether patients received chemotherapy and/or radiation (Supplementary Fig. 1E,F).While there is insufficient sample size in the number of iron supplemented patients to achieve balance on every individual comorbidity, balance was achieved in the Charlson comorbidity index which serves as a general measure of baseline health of the patient cohort 35 .Performing sex-stratified Kaplan-Meier analysis and univariate Cox regression with robust variance estimation on the matched cohorts revealed that iron supplementation was associated with a trend toward prolonged survival in females although failing to reach statistical significance (median-OS 18.6 vs. 12.8 months, P = 0.147, HR 0.61; 95% CI 0.32-1.19)whereas no significant association was found in males after matching (median-OS 13.1 vs. 13.5 months, P = 0.650, HR 0.85; 95% CI 0.41-1.75)(Fig. 4).A, C) and after (B, D) propensity score matching.Anemia, as defined using hematocrit cutoffs of < 36% for females and < 39% for males, was associated with reduced 5-year overall survival.

Male glioblastoma tumors exhibit increased transferrin binding
Since sexual dimorphism was observed for both our analysis on anemia and iron supplementation, we hypothesized that there may be intrinsic differences in transferrin binding capacity between male and female tumors.To assess this, radiolabeled transferrin binding assays were performed on resected patient tumors.Since transferrin binding is tightly regulated by iron content, assessment of transferrin binding capacity of tissues represents a functional measure of tissue iron content 36,37 .Briefly, plasma membrane fractions of resected human glioblastoma samples were isolated and incubated with 125 I-labeled transferrin.We found that plasma membrane fractions from male tumors bound significantly more transferrin per milligram of tissue compared to female tumor samples (Fig. 5).Of note, given that transferrin receptor is also highly expressed on endothelial cells, we cannot rule out that the male tumors may have had increased vascularization compared to the female tumors, however this would also suggest a mechanism where male tumors may have access to more iron 38 .

Discussion
Anemia is a highly prevalent condition that is commonly found in cancer patients and often exacerbated by cancer treatment 2,3 .The role of anemia in glioblastoma patient outcomes is unclear with previous analyses being limited in number and reporting conflicting results.Some studies have found no association between anemia and survival 39 , while others have found a negative association of anemia with survival among glioblastoma patients [40][41][42][43] .These studies are summarized in Table 3 and reviewed in greater detail elsewhere 44 .Of note, these studies have been limited to single institutions and smaller patient cohorts with insufficient size to study sex as a biological variable.Here we report the largest, multi-center, analysis into the association of anemia and survival among adult glioblastoma patients to date.In our analysis of 1346 glioblastoma patients, we found that anemia within the 6 months following diagnosis was associated with reduced survival in male but not in female glioblastoma patients.We further investigated associations of iron supplementation with outcomes among anemic glioblastoma patients and found that supplementation of anemic female patients was associated with a trend toward prolonged survival although failing to reach significance, while no such association was found among anemic male patients.The sexually dimorphic association of anemia with survival in our study raises interesting questions regarding the interplay of sex and tumor biology.Sexual dimorphism in glioblastoma has been well documented with males typically having higher incidence and poorer survival compared to females 1 .The analysis of transferrin binding to male and female tumors identified a potential underlying process contributing to the sexually dimorphic associations of anemia and iron supplementation with survival.Assessment of transferrin-binding capacity represents a functional measure of iron content in tumor tissues.When iron levels are low, transferrin receptor expression is elevated and vice-versa 36,37 .We found that membrane fractions from male tumors bound significantly more transferrin which is consistent with decreased iron stores in male tumors.The elevated transferrin binding in male tumors may represent an attempt by the tumor to increase iron acquisition due to decreased iron stores and is consistent with our observation that anemia seems to have a larger effect on male outcomes compared to female outcomes.We also performed an analysis into associations between iron supplementation of anemic patients and median overall survival.After balancing iron supplemented and non-supplemented cohorts for age at diagnosis, Charlson Comorbidity Index, and whether chemotherapy and radiation were received, we found that iron supplementation in the period surrounding or after glioblastoma diagnosis was associated with a trend toward prolonged survival in females but not in males.Previous studies have suggested that iron supplementation, especially in combination with erythropoietin-stimulating agents, may be an effective approach at managing both absolute iron deficiency anemia as well as chemotherapy-induced functional anemia, but these studies did not investigate sex-differences in the context of glioblastoma 45,46 .Underlying differences in the etiology of the anemia between the sexes in our cohort may also contribute to the sexually dimorphic trends observed in the analysis on iron supplementation.Supporting this theory, we note that in our hemoglobin-stratified analysis, anemic female patients had a lower MCV than non-anemic female patients (Table 1: 89.68 vs. 91.44;P = 0.002) whereas no significant difference in MCV was found in the comparison between anemic versus non-anemic males (Table 2: 90.47 vs. 91.06;P = 0.234), suggesting that iron deficiency may contribute to a larger portion of the anemia observed in the female cohort compared to the male cohort and thus be more amenable to iron supplementation.
Many iron-related disorders including hereditary hemochromatosis and iron deficiency anemia exhibit marked sexual dimorphism in terms of incidence, age of onset, and severity of disease 47,48 .Females are more likely to be anemic compared to males due to physiological blood loss throughout much of adulthood and thus compensatory mechanisms may have evolved due to evolutionary adaptation resulting in sexually dimorphic handling of the downstream effects of anemia, including hypoxic responses 47 .Indeed, studies in animal models examining perinatal hypoxia-induced brain damage have reported that the hypoxia-induced injury was greater in males than in females 49,50 .Pathways activated by hypoxic responses such as those involved in cell death, handling of oxidative stress, and microglial activation have also been reported to exhibit features of sexual dimorphism 51 .
In addition to correcting anemia-induced hypoxic responses, iron itself may potentially contribute to antitumor responses.A study by Anic et al. examined iron content in toenail clippings collected from 193 glioblastoma patients post-diagnosis 52 .While that study did not examine associations between iron content and survival, it did report that toe nail iron appeared to decrease the further it was collected from the date of glioblastoma diagnosis, suggesting that glioblastoma pathology and iron biology may be more closely associated than previously appreciated 52 .Recent work has also demonstrated novel anti-tumor properties of iron including promoting ferroptosis 22 , inhibiting glioblastoma cell migration 23 , and promoting anti-tumor immunity 24 .In addition to differences in the underlying etiology of anemia, the sexual dimorphism observed in trends between iron supplements and survival may, in part, be explained by differences in iron uptake.Previous work in our laboratory demonstrated that, in mouse models, injection of radioactive iron bound to transferrin resulted in significantly higher uptake in female liver and brain compared to male controls suggesting sexually dimorphic regulation of body iron storage and blood-brain-barrier iron transport 53 .
Our study is limited by its retrospective nature and limited sample size for the analysis of iron supplementation of anemic patients.Iron supplements were infrequently utilized in the anemic glioblastoma patient cohort examined here and prospective clinical trials are needed to verify our findings.As these data were largely derived from de-identified electronic health records, data regarding molecular profiling is largely unavailable.Thus we cannot comment on whether differences in molecular profiling may have played a role our findings.In addition, our study lacks an independent validation cohort, yet does include a large sample size as a first investigation to be validated in a future study.Nonetheless, our study brings to attention a potentially clinically significant link between anemia and glioblastoma sex-specific outcomes that should be further investigated with anemia correcting therapies for use among glioblastoma patients to potentially both improve quality of life and provide more favorable outcomes.

Figure 1 .
Figure 1.Anemia status and patient distribution.(A) Median hemoglobin and hematocrit levels within 6 months post glioblastoma diagnosis.(B) Distribution of the 1346 glioblastoma patients by sex, anemia status, and whether the anemic patients received an iron supplement.Created with help from biorender.com.

Figure 3 .
Figure 3. Sex-specific overall survival of patients stratified by anemic versus non-anemic using hematocrit cutoffs.Sex-specific median overall survival was assessed for anemic versus non-anemic patients before (A, C) and after (B, D) propensity score matching.Anemia, as defined using hematocrit cutoffs of < 36% for females and < 39% for males, was associated with reduced 5-year overall survival.

Figure 4 .
Figure 4. Overall survival of patients stratified by iron supplementation status and sex.Median overall survival was assessed for patients that received an iron supplement versus those that did not receive an iron supplement stratified by sex before (A, C) and after (B, D) propensity score matching.Iron supplementation was associated with prolonged overall survival in female patients but not in male patients.

Figure 5 .
Figure 5. Transferrin binding in human glioblastoma samples by sex.Binding of 125 I-tagged transferrin to extracted plasma membrane fractions from homogenized human glioblastoma tumors (n = 6 male, n = 6 female) was assessed by sex.Male tumors had significantly higher binding of radiolabeled transferrin compared to female tumors.P-value corresponds to unpaired two-sample Wilcoxon rank sum test.

matching After matching Hb ≥ 13 g/dL Hb < 13 g/dL P Hb ≥ 13 g/dL Hb < 13 g/dL
Patient samples utilized in this study were retrieved following a Penn State IRB approved protocol with informed patient consent and fully de-identified prior to distribution to the research team.All clinical data used in this manuscript were obtained fully de-identified.This research was conducted in accordance with the Declaration of Helsinki.

Table 3 .
Summary of previous studies examining anemia and glioblastoma with references, year of publication, number of patients in study, and conclusion.